Journal of Open Science Publications Science & Research

Volume 1, Issue 1 - 2014 © Anwar Shahzad 2014 www.opensciencepublications.com An Overview on : A of Woody Medicinal Climbers

Research Article Shiwali Sharma and Anwar Shahzad* Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh-202 002, UP, India *Corresponding author: Anwar Shahzad, Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh-202 002, UP, India, E-mail: [email protected]; [email protected]

Article Information: Submission: 09/01/2014; Accepted: 10/03/2014; Published: 27/03/2014

Abstract Decalepis is one of the most important endangered woody medicinal climbing members of “Periploaceace” family. It comprises five species of twining vines and erect shrubs, D. hamiltonii, D. arayalpathra, D. salicifolia, D. khasiana and D. nervosa. Four of the five species of Decalepis are endemic to the Eastern and Western Ghats of peninsular India; the exception, D. khasiana, is geographically isolated from the peninsular species, occupying forest areas in the Meghalaya state in the easternmost part of India, Bangladesh, Laos, Myanmar and parts of Southern China. D. hamiltonii is the type species and most widespread of the Indian endemics. Three species (D. arayalpathra, D. hamiltonii and D. salicifolia) have clusters of numerous fleshy and tuberous roots with a sweet, vanilla-like fragrance due to the presence of 2-hydroxy-4-methoxy benzaldehyde (2H4MB). The tuberous roots of D. arayalpathra and D. salicifolia are moniliform, while those of D. hamiltonii are cylindrical. The roots of D. khasiana are documented as being non-tuberous, and fragrant due to an isomer of vanillin. Four of the five species ofDecalepis (all except D. nervosa) are utilized in tribal and traditional Indian and Chinese medicine for the treatment of a wide range of ailments including those of the digestive system, lungs and circulatory system. Presently the three peninsular Indian species of Decalepis are threatened in the wild, and listed as endangered (D. hamiltonii) to critically endangered (D. arayalpathra and D. salicifolia) due to over-exploitation and habitat loss. During last few years considerable efforts have been tried to conserve this valuable endangered liana using different strategies of plant tissue culture viz., in vitro shoot regeneration (direct or indirect) and root induction, somatic embryogenesis, hydrogel encapsulation, normal root culture and hairy root culture. The present review provides up-to-date baseline information of all the species of this valuable endangered and endemic medicinal genus for further research work.

Keywords: D. hamiltonii; D. arayalpathra; D. salicifolia; D. khasiana; D. nervosa

Abbreviations Introduction

BA: N-6-Benzyladenine; 13C NMR: Carbon 13 NMR; IAA: Indole- India is one of the 17 megadiversity countries and has all the 13 3-Acetic Acid; IUCN: The International Union for Conservation biomes found in the world, with 3 major hotspots out of a total of 34 [1]. It has been reputed to be a treasure house of a wide range of of Nature and Natural Resources; GA3: Gibberellic Acid; GC: Gas valuable medicinal and aromatic on account of habitant of Chromatography; GC-MS: Gas Chromatography Mass Spectroscopy; divers’ climatic conditions. India harbours about 47,000 species of 2H4MB: 2-Hydroxy-4-Methoxy Benzaldehyde; 2-iP: 2-Isopentyl plants of which 17,000 are angiosperms [1]. The International Union Adenine; Kn: Kinetin; NAA: α-Naphthalene Acetic Acid; PAA: Phenyl for Conservation of Nature and Natural Resources (IUCN) Red List 1 Acetic Acid; PG: Phloroglucinol; H NMR: Proton NMR; AgNO3: of Threatened species include a total of 560 species from India, out of Silver Nitrate; TDZ: Thidiazuron; TLC: Thin Layer Chromatography; which 247 species are in the Threatened category. On a global basis, the TRIA: Triacontanol; Zea: Zeatin IUCN has estimated that about 12.5% of the world’s vascular plants,

01 ISSN: 2349-2805 JOURNAL OF PLANT SCIENCE & RESEARCH Anwar Shahzad totalling about 34,000 species are under varying degree of threat [2]. of only 20 km due to windfall patterns and topography, resulting in However, according to the report of Hamilton [3] an estimated 4,000 the development of small ecological islands with unique floras on the to 10,000 species of medicinal plants face potential local, regional, borders of wet evergreen and dry evergreen and deciduous forests [9]. national or global extinction, with subsequent serious consequences D. nervosa, the northernmost of these narrowly endemic species, for livelihoods, economies and health care systems. is restricted to one of the highest sections of the Western Ghats, Decalepis is one of the most important endangered woody the Nilgiris (Blue Mountains in Tamil), which occupy the western medicinal climbers. This review is aimed at compiling up-to-date corner of Tamil Nadu where it borders Karnataka and Kerala states. information regarding medicinal and economical properties or D. nervosa has been documented at an elevation of ca. 1500 to 2300 related information of all the species of Decalepis over its range of m, on rocky hillsides and exposed areas of deciduous forests near distribution, current status and the role of biotechnology in the Coonoor, Kothagiri, and Wellington. The Nilgiris are geographically conservation of this important genus. isolated from the neighboring mountains to the south by the 32-42 km wide Palghat gap, a low pass in the Western Ghats [4]. Distribution D. salicifolia and D. arayalpathra occupy similar habitats in hilly Decalepis belongs to the family Periploaceace (an offshoot of and mountainous areas to the south of Palghat gap. D. salicifolia has the Asclepiadaceae). It comprises five species of twining vines/erect been documented in three localities, the Anamalai Hills (Kerala and shrubs; Tamil Nadu), Nelliampathy (Kerala) and Marayoor (Kerala) forests. • D. hamiltonii (the type species) The Anamalai Hills (Elephant hills in Tamil Nadu) straddle Kerala and Tamil Nadu states just south of Palghat gap and are known for their • D. arayalpathra abundant wildlife and under consideration by the UNESCO World • D. khasiana Heritage Committee for selection as a World Heritage Site. Further south, D. arayalpathra occupies rocky hill slopes on the Agasthiyar • D. nervosa hills (Kerala) and within Kalakkadu Mandanthurai tiger reserve in • D. salicifolia Tamil Nadu [4]. Four of the five species of Decalepis are endemic to the Eastern D. khasiana was first described from a specimen collected at an and Western Ghats of peninsular India; the exception, D. khasiana, altitude of 900-1200 m, in the Khasia hills in Eastern India. It has is geographically isolated from the peninsular species, occupying since been documented in Laos, on the Tran-Ninh plateau in the area forest areas in the Meghalaya state in the easternmost part of India, of Xieng-Khowang, at an altitude of 800-900 m [10], in the Tamabil- Bangladesh, Laos, Myanmar, and parts of southern China. All five Jaflong hills of the Sylhet district in Bangladesh, and in the Yunnan, species occur in hilly to montane regions, in elevations ranging from Guizhou, and Sicuan provinces of China, in bush land forests and in 100-1200 meters [4]. Myanmar [4]. D. hamiltonii is the type species and most widespread of the Taxonomic History Indian endemics. It is locally called as “Maredukommulu” or The taxonomic history of Decalepis began with the naming of “Nannarikommulu” or “Barresugnadhi” or “Maredugaddalu” D. hamiltonii by Robert Wight and George Arnott Walker-Arnott or “Madinakommulu” in Telugu, “Makaliber” in Kannada, in Wight’s “Contributions to the Botany of India” (1834), based on a “Magalikizhangu” in Tamil and “Swallow root” in English. It grows in specimen collected by Wight in Madras, India [11]. Walker-Arnott, open rocky slopes and crevices of dry and moist deciduous forests of who was collaborating with Wight, upon observing what he referred Karnataka (Hassan, Mysore, Bellary, Tumkur, Kolar), Andhra Pradesh to as “a double row of scales” (referred to in this study as “corona (Kurnool, Chittoor, Nellore, Anantapur, Cuddapah districts) and in lobes” and “interstaminal nectaries”), placed the new species in its Tamil Nadu (Chengalpattu, Coimbatore, Dharampuri, Nilgiri) at an own genus, Decalepis (Greek deca=ten, lepis =scale) [11]. Wight altitude from 300-1200 meters [5]. Though relatively widespread, its mistakenly referred to the specimen in an earlier, unpublished populations are fragmented and gradually declining due to destructive version of his manuscript as being from the herbarium of the Scottish harvesting of the tuberous roots [6]. physician and botanist Francis Hamilton; Walker-Arnott chose to The remaining peninsular species, D. arayalpathra, D. salicifolia honor Hamilton with the specific epithet hamiltonii despite the fact that the specimen was from the Madras Herbarium and not from and D. nervosa, have more restricted distributions, each occupying a Hamilton’s collection [4]. different mountain range in the southern part of the Western Ghats. The south Western Ghats (bordered to the north by the Kalindi river) Decalepis was monotypic until Venter and Verhoeven [12] are extremely diverse floristically, comprising the richest center of synonymized the monotypic Indian genera Baeolepis Decne. ex endemism in peninsular India, with almost 1200 endemic species Moq. and Janakia J. Joseph & V. Chandras. with Decalepis, followed [7,8]. Subramanyan and Nayar [9] compared this assemblage of in 2001 by Utleria Bedd. ex. Benth. & Hook. f., also monotypic hilltops, bounded to the west by the coastal plain and by the semi- and endemic to India, on the basis of “ovate-like corona lobes, an arid Deccan plain to the east, to an island chain in respect to the obconical style-head with flat apex containing translator hollows, distribution of endemic species. In the Western Ghats, the length of and pollen in pollinia” [12,13]. Results of phylogenetic analysis the dry season can range from three to eight months over a distance (excepting D. nervosa, for which molecular data were unavailable)

Citation: Sharma S, Shahzad A. An Overview on Decalepis: A Genus of Woody Medicinal Climbers. J Plant Sci Res. 2014;1(1): 104. 02 JOURNAL OF PLANT SCIENCE & RESEARCH Anwar Shahzad tentatively support this grouping, with the addition of D. khasiana, khasiana are documented as being non-tuberous, and fragrant which Venter and Verhoeven [13] had moved previously from the due to an isomer of vanillin that is also present in the other monotypic Stelmacrypton Baill. To Finlaysonia along with several fragrant-rooted species. No references were found pertaining other monotypic and bitypic Asian genera, on the basis of similarities to the root morphology or chemistry of D. nervosa [4]. in floral form i.e., the gynostegium exposed from a rotate or saucer- ii. Stems: The stems are woody and reddish brown when young, shaped corolla, staminal feet swollen and these topped by corona tend to become purplish when older [12]. Lenticels are lobes and pollen tetrads fused into pollinia (most common on older branches. The stems of D. salicifolia are have pollen in the form of free tetrads). Subsequent molecular studies somewhat succulent, becoming quite wrinkled when dried have demonstrated Finlaysonia sensu [13] to be polyphyletic, and the and the older stems are covered with a grayish wax [4]. characters used to support that grouping to be pleisiomorphic [14]. iii. Leaves: The leaves are opposite, decussate, petiolate and Habit and Habitat exstipulate. In D. salicifolia the leaves are restricted to the ends Three species of Decalepis (D. hamiltonii, D. nervosa and D. of the young branches and clustered at the shoot apices, where khasiana) are scandent to climbing twiners, the commonest growth they sometimes appear to be alternate or whorled due to habit among Periplocoideae [13], while two (D. salicifolia and D. reduced internodes at growing shoot tips. The lamina ranges arayalpathra) are erect shrubs, a condition that has evolved numerous from 2.9-11.5 cm long in all but D. salicifolia, a species having times within the subfamily and is generally correlated with arid leaves that may be up to 19.9 cm long, and narrowly lanceolate, environments or those with seasonal water shortages [15]. Both D. in contrast to the elliptic to ovate or obovate leaves of the other salicifolia and D. arayalpathra are narrow endemics of poor soils species. Leaf apices are acuminate, obtuse or rounded, and in in rocky hill slopes and rock fissures in deciduous and evergreen D. hamiltonii sometimes retuse. The acute to obtuse leaf bases forests, at elevations above 600 m. However, the vining species of in D. nervosa distinguish it from the other species, which Decalepis tend to occupy habitats similar to those of the erect species, have decurrent leaf bases. The lamina is generally flat, but in particularly the narrow endemic D. nervosa. D. hamiltonii it is curved downwards abaxially, particularly at Botanical description the tip, where it is recurved-falcate [4]. iv. Inota (2006) provided a comparative taxonomic detail of all the Inflorescence: The inflorescences are sparsely to multiple five species of Decalepis on the basis of 66 herbarium specimens from branched, few- (up to 10 in D. khasiana and D. arayalpathra; BM, CAL, E, K, LM, MO, NY, P and US [16] (Figure 1); 15 in D. hamiltonii) to many- (up to 50 in D. salicifolia; 80 in D. nervosa) flowered axillary cymes. Branching is monochasial i. Roots: Three species have clusters of numerous, fleshy, throughout in D. hamiltonii, D. arayalpathra and D. salicifolia; tuberous roots with a sweet, vanilla-like fragrance: D. in D. nervosa the inflorescences branch dichasially proximally arayalpathra, D. hamiltonii and D. salicifolia. The tuberous and monochasially distally, and branching is dichasial roots of D. arayalpathra and D. salicifolia are moniliform, throughout in D. khasisana [4]. while those of D. hamiltonii are cylindrical. The roots of D. v. Flower structure and terminology: The flower buds of Decalepis are ovoid; the corollas slightly twisted in bud and with dextrorsely imbricate aestivation. The flowers are perfect, actinomorphic, and pentamerous. D. arayalpathra has the smallest flowers compared to other species, almost 2.5 mm long and 4.0 mm wide at anthesis. The other species have flowers to 3.2 mm long and 5.3 mm wide, with the exception of D. khasiana, which has flowers that are over twice the size of most i.e., 6.5 mm long and 10.0 mm wide [4]. vi. Calyx: The sepals are free, ovate to obovate (oblong-ovate in D. hamiltonii), and 0.7-1.8 mm long by 0.6-1.5 mm wide, with an acute to obtuse (to rounded in D. salicifolia) apex and an irregular to entire, the margin occasionally sparsely ciliate apically (but margin hairs lacking in D. khasiana). The sepals of D. salicifolia flowers are more broadly obovate than those of other species, and thinner, lacking an obvious midrib. Brownish oblong to triangular colleters are located singly or Figure 1: Different plant parts of D. hamiltonii in pairs at the inner bases of overlapping margins of adjacent A. Cylindrical tuberous roots excised from a five year old plant. sepals, but these are occasionally absent in D. hamiltonii [4]. B.Fresh seeds. C.Dry follicle with seeds. vii. Corolla: The corolla is shallowly campanulate (to rotate in D.Seeds with silky hairs. D. khasiana), with ovate to oblong corolla in all species but E.Complete plant of D. hamiltonii planted in loamy soil-6 month old. D. khasiana, with acute to obtuse apices. In most species the

Citation: Sharma S, Shahzad A. An Overview on Decalepis: A Genus of Woody Medicinal Climbers. J Plant Sci Res. 2014;1(1): 104. 03 JOURNAL OF PLANT SCIENCE & RESEARCH Anwar Shahzad

corolla is cream to yellow or greenish, but in D. nervosa and D. ca. 0.2 mm long. In other species the translator receptacles are khasiana the flowers are rose to dark purple. The corolla lobes 0.15-0.25 mm long, the stipes 0.05 to 0.1 mm long [4]. of D. nervosa flowers are thicker than in most species, with xi. Nectaries: In Decalepis the nectaries are located between a tuberculate adaxial surface (smooth in other species); deep adjacent pairs of stamens (interstaminal) and just below grooves radiate from the inner corolla tube adjacent to the them, and in most species they are tabular in form, with base of each corolla lobe, oriented below the pollen translators the exception of D. nervosa, in which they are in the form and extending ca. half way to the lobe apex. Interestingly, of a low ledge. The tabular nectar discs are flattened in cross this species lacks the tubular nectar lobes present in others section and fleshy, and oriented upwards, so that together of the genus, the groove acting in tandem with the shelf-like with the adjacent dilated filament bases (or staminal feet in nectaries to form nectar collecting pockets [4]. D. khasiana), they surround the style branches, causing the viii. Corona: The corona lobes alternate with the corolla lobes, nectar to collect in the upper corolla tube below the pollen emerging just above the filaments and in most cases fused translators, thus attracting pollinators towards a position that laterally to the upper corolla tube to the level of the corolla is favorable for pollen transfer. The ledge-like nectar discs ofD . sinuses, and free above that. In D. hamiltonii, the corona lobes nervosa similarly surround the style (alternating with swollen are free from the corolla tube (i.e., attached only at base). In staminal feet), the nectar collecting in the deep grooves in the D. nervosa, D. salicifolia and D. arayalpathra, the lobes are, corolla that are oriented just below the pollen translators. The respectively, rectangular, oblong or triangular ovate, with tabular nectaries are fleshy and similar in shape to the corona rounded to obtuse apices, and ca. 0.2-0.57 mm long and lobes (but shorter, from 0.2-0.6 mm long) in D. hamiltonii, D. roughly as wide. D. khasiana has triangular lobes about 1.4 salicifolia, and D. arayalpathra. In D. khasiana the lobes are mm long and 0.8 mm wide at the base, with an acute apex, rectangular with rounded edges, in contrast to its triangular and the corona lobes of D. hamiltonii are lanceolate and ca. corona lobes [4]. 1.1 mm long by 0.6 mm wide. The corona lobes of D. nervosa xii. Gynoecium: The ovaries are bicarpellate, subglobose, are uniquely minutely tuberculate abaxially, with an irregular and half-inferior, and vary from 0.8 to 1.2 mm long. The to lobed apex [4]. compound, more or less terete styles are from 0.2-0.3 mm ix. Androecium: The stamens are inserted on the corolla just long (to 0.7 in D. hamiltonii), becoming united and dilated at below the corona lobes, from 0.10 to 0.35 mm (1.0 mm in the apex to form a pentagonal stylar head, which is obscurely D. khasiana) below the corolla sinuses. The anthers are ovate 2-beaked at the center with depressions on the upper surface to oblong-ovate (deltoid in D. nervosa), from 0.5 to 0.6 mm where the pollen translators (a hardened secretion of the stylar long, excluding the connective appendage (1.0 mm long in D. head) are formed. In Periplocoideae the upper surface of the khasiana). The filaments (absent in D. nervosa) are generally stylar head is usually domed and of the species of Decalepis, short (ca. 0.2 mm long) but longer (1.0 mm) in D. khasiana, the stylar head of D. khasiana, which is deltoid in longitudinal and dilated at the base in D. arayalpathra, D. salicifolia, and section, is most typical for the subfamily. In other species of D. hamiltonii (in the last species they are also adnate to the Decalepis, the stylar head is much shallower, in all cases wider corona). Swollen staminal feet are present on the lower corolla than tall. In D. hamiltonii and D. nervosa the upper surface is tube just below the filaments in those species lacking dilated concave, and in D. salicifolia and D. arayalpathra it is broadly filament bases. In most species the stamens are glabrous, convex [4]. but D. khasiana has hairs scattered along the abaxial anther xiii. Fruits: The fruits of Decalepis, like other Periplocoideae, are connective and connective appendage [4]. paired, divaricate follicles. In Decalepis they are more or less x. Pollen and Translators: Pollen is in the form of tetrads cylindrical (D. hamiltonii with a median groove along the that are agglutinated into pollinia at anthesis and deposited suture), narrowing distally towards an acute apex, and 3.5-7.5 onto spoon-shaped translators. The tetrads are elongate and mm long. The endocarp is waxy, bright yellow and ca. 0.2 mm uniplanar, generally elliptic to obovate, and in most species thick. The mesocarp is relatively thin in most species (ca. 0.4 the individual grains of the tetrads are arranged into a mix of mm), but markedly thicker in D. hamiltonii (1.5 mm). The rhomboidal (the most common shape), t-shaped, and linear exocarp is light to dark brown, and in D. hamiltonii and D. forms. In D. nervosa only rhomboidal tetrads were observed. salicifolia extremely waxy, and wrinkled when dry. The fruits D. hamiltonii is unique in having predominantly linear of D. khasiana are not known [4]. tetrads (occasionally rhomboidal). The distal walls of the xiv. Seeds: The seeds are elliptic to obovate and flattened (curved tetrads forming pollinia are in aperturate. Pollen translators along the longitudinal axis towards the funicular side in D. are spoon-like and distally dilated into a broad obcordate nervosa), with a coma of white to yellowish hairs up to 2.9 cm concave receptacle with an emarginate apex. The receptacle long at the micropylar end. Coma hair color varies significantly has a longitudinal groove on the abaxial surface, effectively within species and is not taxonomically informative. The seeds forming two shallow cups in which pollinia are deposited. The are slightly keeled at the point of attachment to the placenta, viscidium is discoid. D. khasiana and D. hamiltonii have the and in D. hamiltonii and D. nervosa the surface is smooth or largest translators, with receptacles 0.6-0.4 mm long and stipes somewhat wrinkled on the funicular side. Testa color ranges

Citation: Sharma S, Shahzad A. An Overview on Decalepis: A Genus of Woody Medicinal Climbers. J Plant Sci Res. 2014;1(1): 104. 04 JOURNAL OF PLANT SCIENCE & RESEARCH Anwar Shahzad

from dark red-maroon to dull black, and varies within the 5-10 cm in diameter and 4-10 roots regenerate from the rootstock. species. Seeds of D. khasiana are not known [4]. A 2-3 year old plant produces 15-20 kg of roots, while one year old plant produces 1-2 kg of roots. Roots are harvested during summer Cultivation months mostly by the Yanadi tribe of Chittoor district and it is the For D. hamiltonii, loamy soils are well suited for the growth main source of income to them until the agricultural works resumes. but soils intermixed with stones encourage the production of long Chemical constituents thick roots. The length and thickness of the roots mostly depends on the soils profile rather than the environment [17]. However the The roots ofD. hamiltonii have been reported to contain aldehyde, information regarding cultivation for other species of Decalepis is not inositols, saponins, amyrins, lupeols [21,22]. Morphologically as well documented elsewhere. well as chemically the plant resembles African liana called Mondia whitei. Both have similar ethno botanical uses and the presence Propagation 2-hydroxy-4-methoxy benzaldehyde (2H4MB), an isomer of vanillin All the species of Decalepis can be propagated from seeds as well as is reported from the volatile oils (96%) [23]. Further GC-MS analysis by means of stem cuttings. For commercial purpose the first method of this oil showed, along with the major component, the presence of is preferred. As the direct sowing of seeds in the field does not give benzaldehyde (0.017%), salicylaldehyde (0.018%), methyl salicylate good results, seedlings are raised in the nursery before transplanting (0.044%), benzyl alcohol (0.016%), 2-phenylethyl alcohol (0.081%), in the field. Collection of seeds from February-March is preferred ethyl salicylate (0.038%), p-anisaldehyde (0.01%), and vanillin to achieve higher viability percentage. Because of short viability, the (0.45%) in minor quantities which are biologically significant [23]. fresh seeds are to be used. The seeds can be raised either in the nursery Hydro-distillation of D. hamiltonii roots yielded an essential oil beds or in polythene bags. Germination starts after about 2 weeks and (0.33% v/w) that contained 2H4MB (37.45%), 2-hydoxybenzaldehyde may continue for another week. Three months old seedlings having (31.01%), 4-O-methylresorcylaldehyde (9.12%) and benzyl alcohol 4-6 leaves are transplanted at a distance of 60 cm × 60 cm in field. (3.16%) as major constituents, with aromatic aldehydes constituting Generally, That sentence will be replaced by this: the crop is cultivated the main fraction of its roots’ essential oil [24]. Thangadurai et al. [25] under rain-fed conditions [17]. Seeds of D. hamiltonii were found to isolated two other compounds from the root extract that potentially germinate soon after early monsoon showers in May on soil floor and account for its insecticidal and anti-microbial properties: α-atlantone rocky areas which are rich in litter and other organic matter. In nature, (2.06% v/w of oil), commonly used to control household insects and seed germination rate is 13% out of which seedling establishment rate nontoxic to mammals, and β-pinene (2.01%), which is used as an is 48%. Thus, the overall seedling establishment rate in relation to insecticide and disinfectant. Whereas, Zhang et al. [26] isolated four total seeds sown is only 6% [18]. Anandalakshmi and Prakash [19] new pregnane glycosides (Stelmatocryptonoside A, B, C and D) from have studied the viability and germination of D. hamiltonii seeds the stems of D. khasiana; and four species of sariva (D. hamiltonii, under in vitro conditions. They have suggested that the use of moist Cryptolepis buchananii, Ichnocarpus frutescens and H. indicus) were filter papers was better substrate for germination (83 to 98%) than shown to have potent anti-inflammatory properties [27]. sand (15 to 28%) after pre-soaking of seeds in hot water (60°C) for 24 A gas chromatographic (GC) procedure was developed by hours. Pretreatment study coupled with microscopic studies indicated Nagarajan and Rao [28] for the assay of 2H4MB in both fresh and that about 14% of seeds were found to be hard seeded. Seeds could dried roots of D. hamiltonii from different origins. They used benzyl tolerate desiccation up to 10% moisture content (65% germination) butyrate as an internal standard. Among the methods tried, steam while nil germination was recorded at 5% seed moisture content. The hydrodistillation was found most suitable for extraction of the volatile seeds were found to be chilling sensitive, thus showing total mortality oils. The quantity of this aromatic compound varied from 0.03 to 0.54% at 10°C. Rapid depletion of seed metabolites is indicative of short (Nagarajan and Rao 2003). In addition 4-hydroxyisopthalic acid, viability. Based on the storage behaviour, D. hamiltonii seeds could 14-aminiotetradecanoic acid, 4-(1-hydroxy-1-methylethyl)-1-methyl- be classified as intermediate seeds. From seed perspective, hard seed 1,2-cylohexane diol, 2-(hydroxymethyl)-3-ethoxybenzaldehyde, coat, need for a well aerated substrate and short viability could be the 2,4,8-trihydroxybicycle (3,2,1) octan-3-one, bis-2,3,4,6-galloyl-α/β- likely impeding factors for inadequate regeneration of the species in D-glucopyranoside, bornerol and ellagic acid have been identified in wild. Saini and Giridhar [20] found that exposing seeds to 0.3% H O 2 2 D. hamiltonii root [29,30]. resulted in effective (94%) seed germination in D. hamiltonii. While Vedavathy (2004) reported 100% germination of immature zygotic Traditional uses embryos when placed on MS medium supplemented with GA (0.05 3 Four of the five species of Decalepis (all except D. nervosa) are ppm), BA (1.0 ppm) and TDZ (1.0 ppm). utilized in tribal and traditional Indian and Chinese medicine for the Harvesting treatment of a wide range of ailments including those of the digestive system, lungs and circulatory system [31-33,26]. The principal organs The roots ofD. hamiltonii come to maturity in about 12-14 months used are the tuberous roots, which are fragrant and sweet tasting after planting depending upon the soil and climatic conditions. The (fragrant but non-tuberous in D. khasiana), with a vanilla-like taste roots can easily be harvested by digging around the plant leaving the and odor [34,33,28,35]. D. hamiltonii, the type species, is the most central core of the root and stem for regeneration. Some plants could widespread and utilized species in the genus. be used to harvest roots up to 4-5 years, as vigorous regeneration and growth resumed even with a single left over root. Each root is The roots of D. hamiltonii are also used in folk medicine and in

Citation: Sharma S, Shahzad A. An Overview on Decalepis: A Genus of Woody Medicinal Climbers. J Plant Sci Res. 2014;1(1): 104. 05 JOURNAL OF PLANT SCIENCE & RESEARCH Anwar Shahzad

Ayurvedic preparations [36]. Dried roots are available in Indian drug extract of D. hamiltonii was evaluated at different concentrations by stores and local tribal markets. Ancient tribes in the Western Ghats poisoned food technique against 8 species of Fusarium, 10 species of India use its roots particularly for inflammation [37]. These people of Aspergillus, 3 species of Penicillium, 2 species of Drechslera and procure and habitually carry the roots with them and chew the same Alternaria alternata. [43]. Mohana et al. [44] found that the antifungal whenever the digestion may seek relief. Besides treating indigestion, active principle is a phenolic compound, 2H4MB. In vitro antifungal the roots have also been used locally to stimulate the appetite and to activity assay of different concentrations of 2H4MB isolated from D. relieve flatulence and to act as a general tonic [17]. Tuberous roots are hamiltonii against 6 important seed-borne fungal pathogens, namely, used as a flavoring principle [21] and a blood purifier [31,38]. Roots A. alternata, Drechslera tetramera, Fusarium oxysporum, Fusarium are used to cure dysentery, cough, bronchitis, leucorrhoea, uterine proliferatum, Pyricularia oryzae and Trichoconis padwickii isolated hemorrhage, skin disease, fever, indigestion, vomiting, chronic from paddy seeds revealed that, the compound 2H4MB showed rheumatism, anemia and blood diseases. They are also consumed as significant antifungal activity. Among the fungi tested, F. proliferatum pickles and as a popular cool drink in the forest areas of the Eastern and showed the highest inhibitory activity, whereas P. oryzae showed least Western Ghats, known as Nannari which has a cooling effect without inhibitory activity [44]. any toxic effects on human beings [39,17,40]. It finds its use as culinary ii. Antibacterial activity: Thangavel et al. [45] studied spice because of its high priced aromatic roots and marketed on a antibacterial potential of crude petroleum ether extract of leaf callus large scale [18]. Root extract can be used not only as food preservative tissue of D. hamiltonii against 5 bacterial pathogens using agar well (to replace for the toxic butylatedhydoxy anisole and butylatedhydoxy diffusion method. Among the tested bacterial strains, inhibitory toluene currently under use) but also can be used in the preparation activity of the callus extract was minimum against Klebsiella of neutraceuticals and pharmaceutical products [41]. The roots of D. pneumoniae (5 mm) and followed by Proteus vulgaris (6 mm). hamiltonii are little bitter and then sweet. It is so characteristic with a Maximum inhibitory activity was observed against Salmonella typhi familiar lingering after taste and smell of vanillin, the substance that is (11 mm). From this observation it was evident that phytochemical in Vanilla planifolia, an orchid used in ice-creams, chocolates, drinks principles which are responsible for the curative (antibacterial) etc. Although vanillin has been synthesized since 1874 natural source activity of D. hamiltonii could have a constant expression pattern of this flavoring are still in demand and the roots of Decalepis species in a specialized set of cells even after their rapid division. Later on can be used as substitute for vanillin. Devi and Latha [46] also reported antibacterial activity of various D. arayalpathra was discovered relatively recently, and described root extracts of D. hamiltonii against E. coli, K. pneumoniae, S. typhi, as Janakia arayalpathra [42]. Pushpangadan et al. [31] discovered Proteus mirabilis, Vibrio cholerae, Shigella sonnie, Serritias species, S. during an ethno-medico-botanical investigation that though relatively aureus and Bacillus subtilis by disc diffusion method. new to science, D. araylpathra has long been recognized by the Kani iii. Insecticidal activity tribe of Kerala, they consider it an important medicinal plant, as reflected in the vernacular name ‘amrithapala’, which translates as Several studies have been conducted to evaluate the insecticidal ‘the plant which gives the milky ambrosia or nectar of immortality’ activity of D. hamiltonii against 3 coleopteran stored product pests, (amritha=ambrosia or ‘nectar of immortality’, pala=milk, likely viz., rice weevil, the lesser grain borer and red-rust flour beetle [24,47]. referring to the abundant white latex present in all plant parts). They Further, the residual deposits of 2H4MB has been assayed for contact make use of juice obtained from the tuber as a remedy for peptic toxicity on rice weevil (Sitophilus oryzae), Rhyzopartha dominica and ulcer, as a rejuvenating tonic, and to cure for external cancers [32]. Tribolium castaneum. Thus, the aromatic chemicals of D. hamiltonii In all other species, D. salicifolia has been known to science since its have been established with insecticidal and pesticidal properties. description in the 19th century; its ethnobotanical use by the semi- iv. Anti-oxidant activity: Free radicals are implicated in the nomadic Kadars, Malasars, and Muthuvans tribes was discovered only etiology of several diseases, such as cancer, antherosclerosis, diabetes, recently. The tubers of ‘Mahali kizhangu’ (Mahalekshmy=goddess of neurodegenerative disorder and aging. Antioxidants prevent the wealth, kizhangu=root tubers) are hung from the front of huts to bring damage to macromolecules and cells by interfering with the free good fortune, and pickles made from the roots are used to treat colic, radicals. Natural products containing antioxidants from plants bleeding ulcer, tuberculosis, asthma, and skin diseases [33]. Similarly, are believed to modulate oxidative stress and to prevent or delay all parts of D. khasiana including its fragrant, vanilla-scented roots degenerative disorders [48]. Harish et al. [29] characterized 6 pure are used in Chinese folk medicine, for the treatment of bronchitis, compounds with strong antioxidant properties from the methanolic cough, stomach and rheumatic aches, and influenza. No records of extract of D. hamiltonii roots by using MS, 1H NMR, 13C NMR and ethnobotanical use were found for D. nervosa. two-dimensional NMR spectroscopic techniques and including a Medicinal Importance novel antioxidant molecule, bis-2,3,4,6-galloyl-α/β D glucopyranoside (aptly named decalepin) from D. hamiltonii root extracts. Srivastava Numerous recent investigations have tested the medicinal et al. [29] also reported antioxidant potential of the methanolic and properties of the different species of Decalepis supported the veracity aqueous extracts of D. hamiltonii roots. Murthy et al. [49] found that of various traditional ethnomedicinal claims. As compared to other antioxidant activity was associated with 2H4MB. They subjected species, D. hamiltonii has been much evaluated for its medicinal different parts of aromatic roots viz., whole tuber, peel, tuber without properties as below; peel and medullary portion. Maximum antioxidant activity was i. Anti fungal activity: The antifungal activity of aqueous found with peel extract followed by medullary Portion [49]. Later on,

Citation: Sharma S, Shahzad A. An Overview on Decalepis: A Genus of Woody Medicinal Climbers. J Plant Sci Res. 2014;1(1): 104. 06 JOURNAL OF PLANT SCIENCE & RESEARCH Anwar Shahzad

Srivastava et al. [50] isolated another antioxidant compound from the tumor cell counts and increased median survival time (MST). EAT aqueous extract of the roots of D. hamiltonii and identified it as ellagic cells induced alterations in hematological profile and the serum marker acid (EA), based on NMR and MS studies.Anti-typhoid activity enzymes aspartate abd alanine transaminases (AST, ALT), alkine Kumuda et al. [51] studied in vitro anti-typhoid activity of various phosphatase (ALP) and lactate dehyrogenase (LDH) were restored by roots extracts of D. hamiltonii. The powdered roots were extracted DHA treatment. The antitumor effect of DHA was comparable to that with various solvents viz., petroleum ether, chloroform, and ethyl of the anticancer drug cyclophosphamide suggesting the anticancer acetate by Soxhlet extraction method and the extracts were tested on potential of the root extract of D. hamiltoniii [53]. typhoid causing organisms. Among these, extracts of petroleum ether Causes of its Extinction and Need of Micropropagation and chloroform showed a significant activity against S. Typhi (ATCC 14028), S. paratyphi A and S. paratyphi B respectively on comparison In traditional Indian medicine, an estimated 90% of the species with Ciprofloxacin [51]. used for plant-based medicines are collected from wild populations, and roots are the plant organ most commonly used, resulting in v. Ulcer preventive activity destructive harvest since the whole plant must be pulled from Srikanta [30] reported a pectic polysaccharide from D. hamiltonii the ground [35]. Presently the three peninsular Indian species of roots containing a sulphonamide group and phenolics as an effective Decalepis are threatened in the wild, and listed by IUCN (International antiulcer compound in vivo. They envisaged a multi-potent role for Union for Conservation of Nature) as endangered (D. hamiltonii) to this phenolic polysaccharide in the upregulation of mucin, antioxidant critically endangered (D. arayalpathra and D. salicifolia) due to over- levels, modulation of oxidative status, inhibition of H+, K+-ATPase exploitation and habitat loss. Destructive harvesting of the narrow activity against swim and ethanol stress induced ulcers in experimental endemics D. arayalpathra and D. salicifolia, particularly vulnerable animal models, in addition to its ability to inhibit Helicobacter pylori. given their limited distributions, is escalating as their medicinal Roots containing phenolics at 0.12 g GAE/g, prevented stress-induced properties become known [54] and even the relatively widespread D. gastric ulcers in animal models by 80-85%. Histopathological analysis hamiltonii faces threats due to its increasing use as a substitute for the revealed protection to the disrupted gastric mucosal layer and important Ayurvedic medicinal plant [55]. In D. + + epithelial glands. SRPP also inhibited H , K -ATPase in vitro, at an hamiltonii, extended flowering pattern, self-incompatibility, pollinator IC50 of 77 μg/mL as opposed to that of 19.3 μg/mL of Lansoprazole limitation, absence of seed dormancy, abortion of a considerable and H. pylori growth at Minimum Inhibitory Concentration (MIC) of percentage of seedlings prior to establishment are contributing 150 μg/mL. In addition, free radical scavenging (IC50-40 μg/mL) and factors for the regulation of its population size [56]. Moreover, reducing power (3200 U/g) activities were also observed. They also the absence of any organized cultivation of this plant [57] calls for concluded that SRPP is non-toxic as opposed to other known anti- immediate conservation measures. Recent efforts aimed at meeting ulcer drugs, and therefore may be employed as a potential alternative the growing demand for these plants and their useful products while for ulcer management. at the same time ensuring the protection of wild populations include vi. Anti-diabetic activity: Sumalatha et al. [52] evaluated the clonal propagation techniques using nodal/axillary bud explants, anti-diabetic activity of extracts of the D. hamiltonii in alloxan induced which have been developed and optimized for each of the three diabetic rats. The fall of blood glucose levels after administration of ethnomedically important peninsular species [55,58-61,54]. The aqueous, methanol and petroleum ether extracts at a dose of 200 mg/ Foundation for Revitalization of Local Health Traditions (FRLHT), kg body weight were found to be 69.43, 62.04, and 49.61%, respectively, a non-governmental organization based in southern India, has been after 4 hours of oral administration. At the same dose the acute oral monitoring populations of D. hamiltonii and established Medicinal administration of aqueous extract showed significant decrease of Plant Conservation Areas focused on the in situ conservation of D. blood glucose loaded normoglycemic rats. In vitro glucose uptake arayalpathra and D. salicifolia. studies suggest that D. hamiltonii root extracts has direct insulin like Role of Biotechnological Tools for ex vitro Conservation effect which can enhance the peripheral utilization of glucose by rat hemi diaphragm. In vitro propagation of rare and threatened plants is generally undertaken to multiply and conserve the germplasm especially vii. Antitumor activity: Cancer is a major aliment that affects when population numbers are low in wild, difficult to regenerate by several organs and chemothaerapy is widely practiced to treat the conventional methods and where population has decreased due to disease. Chemotherapy with or without radiation, although effective over exploitation by destructive harvesting. The modern technology of for many cancers, is accompanied by severe side effects due to the micropropagation provides numerous advantages over conventional toxicity of the anticancer drugs. Several anticancer drugs are derived propagation methods like mass production of true-to-type and disease from plants such as taxol, cycolphosphamide, vincristine, vinblastine free plants of elite species in a highly speedy manner irrespective of and there is great interest in the search for newer and safer drugs from the season within smaller space and tissue source. During last few plants. In this regard, Zarei and Shivanandappa [53] have shown the years considerable efforts have been tried to conserve this valuable antitumor effect of D. hamiltonii root extract (DHA) against Ehrlich endangered liana (Table 1). Following strategies of plant tissue culture Ascites Tumor (EAT) cells in mice and compared its effect with that of have been used for efficient propagation and conservation; the anticancer drug, cyclophosphamide (CP). Treatment of EAT cell bearing mice with aqueous extract of D. hamiltonii (50 mg/kg and 100 i. In vitro shoot regeneration (direct or indirect) and root mg/kg b.w.) showed a significant reduction in ascites tumor volume, induction

Citation: Sharma S, Shahzad A. An Overview on Decalepis: A Genus of Woody Medicinal Climbers. J Plant Sci Res. 2014;1(1): 104. 07 JOURNAL OF PLANT SCIENCE & RESEARCH Anwar Shahzad

Table 1: In vitro shoot regeneraion using different explants of D. hamiltonii (chronological order). Explant Mean no. of shoots/ Mean shoot length Mode of regeneration Treatment References Type explant (cm) L Indirect BA (1.9 mg L-1) + NAA (0.05 mg L-1) 4.4 7.2 [62] Direct NS BA (2.0 mg L-1) + NAA (0.5 mg L-1) 12.8 5.8 [63]

NS Direct BA (17.76 µM) + NAA (0.53 µM) 4 - [64]

ST Direct TRIA (20 µg/l) >5 - [57]

NS Direct BA (31.08 µM) + PAA (14.68 µM) 6.4 2.9 [65]

L Somatic embryogenesis BA (10.65 µM) + zea (9.12 µM) 8 (Somatic embryos) - [69]

NS Direct BA (1.1 µM) + PG (800 µM/l) + GA3 (5.8 µM) 4.0 3-5 [66] ST Direct 2-iP (4.9 µM) 6.5 - [60] L-leaf, ST-shoot tip, NS-nodal segment, BA-N-6-benzyladenine, IAA-Indole-3-acetic acid, NAA-α-naphthalene acetic acid, PAA-Phenyl acetic acid, PG-Phloroglucinol,

GA3-Gibberellic acid, TRIA-triacontanol, zea-Zeatin, “-”-data were not provided in the respective study.

ii. Somatic embryogenesis synergistic effect on shoot multiplication though nodal segments when added with BA and GA . Maximum number of shoots per iii. Hydrogel encapsulation 3 culture was observed on MS medium containing 1.1 µM BA, 5.8 µM iv. Normal root culture GA3 and 800 µM PG. Subculturing of the shoots onto MS medium containing optimum concentration of BA (5.6 µM), PG (200 µM) and v. Hairy root culture TRIA (0.11 µM) produced elongated shoots along with secondary In vitro shoot regeneration (direct or indirect) and root shoot formation. Later on, Giridhar et al. [67] compared the influence induction of different cytokinins (2-iP, BA, Kn, TDZ and zea) on shoot D. hamiltonii: For the first time, George et al. [62] studied in multiplication through shoot tips. Maximum number of multiple vitro growth and development of multiple shoots from leaf calli of shoots (6.5) was noticed on MS medium supplemented with 4.9 µM D. hamiltonii. They used “Response Surface Methodology” (RSM) 2-iP. Further elongation of shoots and adventitious shoot formation to evaluate the effect of BA, NAA and sucrose on multiple shoot was obtained on MS medium with 2.5 µM 2-iP and 0.3 µM GA3 (Table induction and to develop an efficient micropropagation system. The 1). optimum combination for maximum shoot regeneration (4.4 shoots) Giridhar et al. [60] reported the improvement of 2H4MB of -1 -1 was 1.9 mg L BA and 0.05 mg L NAA. Shoot number exhibited a micropropgated plants under TRIA treatment. The microppropgated positive linear relationship with BA concentration and was maximum plants were raised from nodal explants on MS medium supplemented at the highest levels of BA, while sucrose had no significant effect. with 2.0 mg/l and 0.5 mg/l NAA in stage I and MS medium However, maximum shoot length was observed when sucrose supplemented with 2.0 mg/l IBA in stage II by following the method concentration was maximum (3%) in the optimal medium. Bais reported earlier by Bais et al. [63]. They were then hardened in a et al. [63] used axillary bud for micropropagation and achieved green house for 2 months. These micropraogated plants of 12-15 maximum shoot number (12.8 shoots per culture), shoot length (5.8 cm shoot length were used in experiments. They observed improved cm) and number of leaves (16.4) on MS medium supplemented with plant growth and yield of tubers with enhanced flavor compound, by BA (2.0 mg L-1) and NAA (0.5 mg L-1) after 30 days of incubation. using TRIA soil drenching. The administration of TRIA (0.228 µM) Similarly, Anitha and Pullaiah [64] used nodal segments and reported soil drenching twice by giving second treatment with a gap of 60 days maximum of 4 shoots per explant on MS medium supplemented with after initial treatment, showed fast sign of growth and good yield of 17.76 µM BA and 0.53 µM NAA. Reddy et al. [57] studied the effect of tubes with enhanced flavor content. The GC-MS profile of 2H4MB triacontanol (TRIA) on shoot multiplication of in vitro derived shoot in tubers showed significant improvement over controls (1.5 times tips. Among different concentrations (2-20 µg -1L ) of TRIA used, 20 more), which could be useful for various commercial application. µg L-1 TRIA was found to be most effective for shoot proliferation. While Giridhar et al. [65] found that the addition of phenylacetice Giridhar et al. [68] analysed the production of 2H4MB in roots acid (PAA) had a significant effect on shoot multiplication through of tissue culture raised and acclimatized plants of D. hamiltonii. They nodal segments. The maximum number of shoots per culture (6.4 used axillary buds excised from field grown plants to initiate multiple shoots) was produced on MS medium supplemented with 31.08 µM shoots on MS medium supplemented with 2 mg/l BA and 0.5 mg/l BA and 14.68 µM PAA, while the longest shoot length (4.5 cm) and IAA. Profuse rooting was achieved when actively growing shoots nodes were obtained on MS medium supplemented with 22.2 µM BA were cultured on MS medium supplemented with 1.0 mg/l IBA. and 14.68 µM PAA. Shoot subcultured on MS medium supplemented Regenerated plants were grown successfully in the plains, in contrast with 22.2 µM BA and 14.68 µM PAA elongated along with secondary to wild growth in high altitudes and rocky crevices of hilly regions. shoot formation. Roots of different sizes from one-year-old tissue culture raised field Similarly Gururaj et al. [66] found that phloroglucinol (PG) had grown plants had the same profile of 2H4MB as that of wild plants. A

Citation: Sharma S, Shahzad A. An Overview on Decalepis: A Genus of Woody Medicinal Climbers. J Plant Sci Res. 2014;1(1): 104. 08 JOURNAL OF PLANT SCIENCE & RESEARCH Anwar Shahzad maximum of 0.14% and 0.12% 2H4MB was produced in roots of one micropropagation of D. arayalpathra in the year 2005. Gangaprasad year old tissue culture derived plants and greenhouse grown plants et al. [59] cultured single node from young top shoots on MS medium respectively. supplemented with 0.1 to 5 mg/l BA. All the concentrations of BA induced single axillary shoots of varying length. However, MS medium The success of in vitro regeneration relies on an efficient rooting supplemented with 0.1 mg/l BA supported rapid growth and produced in regenerated shoots and their subsequent acclimatization which is the longest shoots (6.8 cm) in 60 days. For further multiplication, the often problematic in some plant species. Bais et al. [63] investigated nodes and shoot tips from in vitro derived shoots were re-cultured in vitro rooting of D. hamiltonii by using silver nitrate (AgNO ). They 3 on MS medium fortified with 0.5 mg/l BA which produced 8 cm long found that medium comprising of MS salts with IAA (2.88 μM), shoot having 5-7 nodes in 30 days period. Further, the top microshoot induced poor rooting and root emergence did not occur until after cuttings (3-5 cm) with 2-3 nodes, subcultured onto MS medium 25 days. The resultant roots were stunted. However, the addition supplemented with 1.5 mg/l IAA, produced an average of 6.3 roots in of 40 μM AgNO improved root initiation and elongation. The 3 30 days period. The rooted plants after hardening were reintroduced promotive effects of AgNO on rooting may result from inhibition of 3 into their natural habitat at Kallar reserve forest, Thiruvananthapuram. ethylene action. It was noticed that the addition of ethephon to the After 2 years, 84% survival of reintroduced plants was recorded. rooting medium, excessive callusing was observed in explants in all In another study of Sudha et al. [54] it was suggested that nodal the treatments. Addition of 40 μM AgNO3 to ethephon containing medium resulted in improvement in root initiation and elongation. explants of greenhouse-raised plants were more desirable than Ethylene production was monitored in all the treatments with IAA/ cotyledonary nodal explants of aseptic seedlings of D. arayalpathra. The basal nodes (73%) of 12-16-week-old greenhouse-grown plants AgNO3/ethephon and it was observed that the treatment with IAA (2.88 μM) alone showed a greater increase in ethylene production cultured in MS medium containing 12.96 µM BA, 2.48 µM 2-iP and 2.68 µM NAA formed 16-17 cm long unbranched robust solitary when compared with AgNO3, ethephon and their combinations. shoots in 8 weeks. Cotyledonary nodal explants cultured in the same For efficient rooting of in vitro-derived microshoots by using medium showed multiple shoot formation and axillary branching. But phloroglucinol (PG), activated charcoal and CoCl2 was investigated the shoots were thin, fragile and not suitable for mass propagation. by Reddy et al. [58]. Transfer of in vitro-derived shoots to MS medium Single nodes of a solitary shoot subcultured on MS medium supplemented with 8.8 µM IBA and 1.43 µM IAA resulted in root containing 2.22 µM BA and 0.24 µM 2-ip together produced 9.8 nodes induction. IBA was found to be a good root-promoting agent, dipping from 18.0 cm long shoots within 5-6 weeks. The basal nodes of the of explants in 4.4 μM IBA for 30 min and subsequent inoculation on shoots so formed were repeatedly subcultured to increase the stock of MS basal medium was also beneficial for root induction Reddy et al. propagules while 2.5-3.0 cm terminal cuttings were used for rooting. [58]. While, Anitha and Pullaiah [64] reported a maximum of 2 to 3 The best root induction (68%) and survival (86%) was achieved on roots on MS medium containing 16.11 μM NAA. Whereas Reddy et half-strength MS medium supplemented with 1.07 µM NAA. Field- al. [57] found that TRIA at 5 and 10 μg played an effective role in root established plants showed uniform growth and phenotypic similarity induction in D. hamiltonii. Giridhar et al. [65] found that the shoots to parental stock [54]. rooted on medium containing 9.8 µM IBA showed 80-90% survival Somatic Embryogenesis: As compared to the other species of rate under field conditions. While Gururaj et al. [66] found best Decalepis, somatic embryogenesis is only reported in D. hamiltonii rooting on 5.38 µM NAA and 400 µM PG containing MS medium. through leaf culture by Giridhar et al. [69]. Nodular embryogenic The efficient root formation was achieved when the elongated shoots callus was developed from the cut end of the explants on MS medium of D. hamiltonii inoculated on medium supplemented with various containing 2, 4-D and BA. On subsequent transfer of explants with phenolic compounds along with 9.8 µM IBA within 5-6 wk [65]. primary callus on to the MS medium containing zea (13.68 µM) and They found that PG and salicylic acid interaction with IBA stimulated BA (10.65 µM) resulted in the differentiation of somatic embryos maximum in vitro rooting of shoots (Table 2). directly from nodular tissue. The maturation of embryos took place D. arayalpathra: There are only two reports for the on the same medium. The efficiency of somatic embryogenesis may

Table 2: Effect of different treatments on in vitro root induction of microshoots of D. hamiltonii (chronological order).

Treatment Rooting Response (%) Field Survival (%) References

MS medium + IAA (0.5 mg/l) + AgNO3 (40 µM) 89.6 80-90 [55,63] MS medium + IBA (4.4 µM) + PG (69 µM) + activated charcoal (0.25%) 80.0 75 [57]

MS medium + IBA (4.4 µM) 100 80 [58]

MS medium + TRIA (5 µg/l) - 70 [57]

MS medium + NAA (5.38 µM) + PG (400 µM) - 80-90 [66]

MS medium + IBA (1 mg/l) - - [69]

MS medium + IBA (9.8 µM) + PG (10-4 M) 100 90 [60]

IAA- Indole-3-acetic acid, IBA- Indole-3-butyric acid, NAA- α-Naphthalene acetic acid, PG- Phloroglucinol, TRIA- Triacontanol, AgNO3-Silver Nitrate, CoCl2-Cobalt chloride, “-”-data were not provided in the respective study.

Citation: Sharma S, Shahzad A. An Overview on Decalepis: A Genus of Woody Medicinal Climbers. J Plant Sci Res. 2014;1(1): 104. 09 JOURNAL OF PLANT SCIENCE & RESEARCH Anwar Shahzad enhanced when the embyrogenic calli with somatic embryos were normal root culture of D. arayalpathra from leaf and inter nodal subcultured onto MS basal medium supplemented with 4.56 µM explants of in vitro-raised shoot cultures in MS medium containing zea and 10.65 µM BA. The mature embryos developed into complete 2.5 mg/l BA, 0.5 mg/l 2-iP and 0.5 mg/l NAA. Shoot cultures were plantlets on growth regulator-free MS medium. maintained on MS agar medium supplemented with 0.5 mg/l BA and 0.05 mg/l 2-iP or and 0.05 mg/l NAA and sub-cultured at 5 weeks Hydrogel Encapsulation: Encapsulation technology represents interval. Leaf explants incubated in total darkness on half-strength a new and powerful tool in the plant nursery field as well as in MS medium supplemented with 0.5 mg/l IBA and 0.2 mg/l NAA approaches to germplasm conservation and exchange of plant favoured induction of roots (5.820 g). A 100 mg fresh root tissue materials between laboratories [70]. Previously, synthetic seed or cultured in 80 ml half-strength MS liquid medium supplemented with artificial seed (or synseed), described as ‘‘artificially encapsulated 0.2 mg/l IBA and 0.1 mg/l NAA, under continuous agitation (80 rpm), somatic embryos (bipolar structure) which can be used for sowing yielded 3.433 g fresh and 0.734 g dry weight of roots. Roots grown in under in vitro or ex vitro conditions’’. But due to low success and high this optimal medium produced maximum content of 2H4MB (0.16%) cost of somatic embryo production, shoot tips, nodes, bulbs or other after 6 weeks of culture. The root cultures were maintained up to the meristematic tissue (unipolar structures) that can produce a whole 7th passage without decline of growth [35]. plant may also be encapsulated which are also considered as synthetic seeds [71]. A wide range of woody plants has been re-grown from Hairy root culture encapsulated shoot tips or nodes [72,73]. The normal root culture system was slow growing with a short An efficient protocol has also been developed for short-term life-span, as mentioned in an extensive review [76]. Advances in storage and conservation of D. hamiltonii using encapsulated plant cell and tissue culture, combined with improvement in genetic nodal segments [74]. Only juvenile nodal segments, excised from engineering, specifically transformation technology has opened aseptic seedlings were used for encapsulation. It was found that the new avenues for high volume production of pharmaceuticals, encapsulation significantly affected by the concentrations of sodium nutraceuticals, and other beneficial substances [77]. Transgenic hairy root cultures have revolutionized the role of plant tissue culture in alginate (Na-alginate) and calcium chloride (CaCl2·2H2O). A gelling secondary metabolite production from the wild and commercial matrix of 4% Na-alginate and 100 mM CaCl2·2H2O was most suitable for the production of ideal Ca-alginate beads. Maximum shoot re- medicinal plants. Hairy root culture is an attractive experimental growth (77.00%) was recorded on MS basal medium supplemented system, as they are long-term aseptic root clones, genetically stable with 5.0 µM BA, 0.5 µM IAA and 30.0 µM adenine-sulphate (ADS) with growth are comparable to those of the fastest-growing cell after 6 weeks of culture. Microshoots, recovered from encapsulated suspension culture [75]. This approach has been successfully applied nodal segments (capsule) were best rooted on half-strength MS for D. hamiltonii and D. arayalpathra as below; medium containing 2.5 µM NAA. Capsules of D. hamiltonii could be D. hamiltonii: Samydurai et al. [78] reported Agrobacterium stored up to 8 weeks at low temperature (4°C) [75]. rhizogenes mediated hairy root culture and genetic transformation Being small in size the capsule, therefore can provide an effective of D. hamiltonii. They have reported the total dry matter content tool for storage and exchange of this endemic and endangered plant of 2H4MB by hairy root culture of D. hamiltonii from internode species, potentially overcoming many of the difficulties associated and roots explants inoculated with A. rhizogenes. The addition of with long-distance transport of plant germplasm. However, in this acetosyringone to basal MS liquid medium fortified with NAA and protocol rooting could not be possible in a single step on re-growth IBA considerably enhanced the hairy root production. PCR analysis media which calls for further refinement in the established protocol of Ri plasmid on transformed internode and root tissues showed DNA in such a way that rooting could be achieved in a single step (on re- amplification at 780 bp amplified in internode, root derived hairy growth-medium). roots. The highest biomass of dry matter content (25.36 and 24.12 g) was observed in liquid MS medium containing 0.3 mg/L NAA, co- Similar to the somatic embryogenesis, exploitation of cultivated with A. rhizogenes on the internode and root explants. After encapsulation technique for the conservation of other species of that the internode and root explants were cultured on MS medium Decalepis is still needed for their successful reintroduction in natural supplemented with 0.3 mg/L IBA thereby exhibited improved biomass habitat. content of dry matter 23.56 and 22.91 g respectively. The dry matter content of biomass was extracted with 1 mg/ml methanol to measure Normal root culture the total content accumulation of 2H4MB in hairy roots cultures by D. hamiltonii: Giridhar et al. [60] studied the production using HPLC [77]. of 2H4MB by normal root cultures of D. hamiltonii. The flavour compound was extracted with dichloromethane, evaporated to dryness D. arayalpathra: Sudha et al. [79] studied A. rhizogenes mediated and dissolved in ethanol for qualitative (TLC) and quantitative (GC- transformation of D. arayalpathra by infecting juvenile hypocotyl and cotyledon explants with different strains, including A4, MTCC MS) analysis. Maximum root biomass and the maximum content of 532, TR105 and LBA 5402. Hypocotyl explants induced hairy roots flavour compound (40 μgg-1 dry weight) were recorded after 45 days at a higher frequency (53.2 ± 0.3 %) than cotyledons (32.1 ± 0.2 %) of growth on Murashige and Skoog (MS) medium containing 1.0 when infected with the most virulent strain TR105. The explants mgL-1α-naphthaleneacetic acid (NAA). co-cultivated 48 h in half-strength salts and vitamins of MS basal D. arayalpathra: Sudha and Seeni [35] reported for fast-growing medium (half-MS medium) induced hairy roots either directly from

Citation: Sharma S, Shahzad A. An Overview on Decalepis: A Genus of Woody Medicinal Climbers. J Plant Sci Res. 2014;1(1): 104. 010 JOURNAL OF PLANT SCIENCE & RESEARCH Anwar Shahzad the wounds or followed by the formation of gall like structures. 5. Anonymous (2003) The Wealth of India: A Dictionary of Indian Raw Materials Irrespective of the explants, the strain MTCC 532 induced callus and Industrial Products. CSIR, New Delhi.Vol 3: 24. alone. The root initials on the galls proliferated vigorously and 6. Giridhar P, Gururaj B and Ravishankar GA (2005) In vitro shoot multiplication elongated more rapidly when they were segmented and subcultured through shoot tip cultures of Wight & Arn., a threatened on half-MS medium than the proliferation and elongation of directly plant endemic to southern India. In Vitro Cell Dev Biol Plant 41: 77-80. emerged roots. The established hairy roots showed intermittent gall 7. Nayar MP (1989) In situ conservation of wild flora resources. Bull Bot Surv formation which was the active sites for hairy roots induction. The India 29: 319-333. molecular evidence of rol A and rol C gene integration was confirmed 8. Nayar MP (1996) Hot spots of endemic plants of India, Nepal and Bhutan. by PCR amplification and southern blot hybridization. Growth Tropical Botanical Garden Publishers, Thiruvananthapuram. of the hairy roots was undertaken by measuring root growth unit 9. Subramanyan K, Nayar MP (1974) Vegetation and phytogeography of the after culturing root tips in half-MS medium and determined fresh Western Ghats. Ecology and Biogeography of India. 23:178-196. weight/dry weight/conductivity during time-course study in shake 10. Thénint A (1936) Un nouveau genre d’Apocynacées. Bull Soc Bot France flask cultures. The maximum biomass and accumulation of the root 83: 389-393. specific compound, 2H4MB (0.22 % dry weight), was recorded at the 11. Noltie HJ (2005) The botany of Robert Wight (Regnum vegetabile 145). 6th week of growth, which was more than that observed in normal root Gartner Verlag, Liechtenstein. cultures (0.16 % dry weight) [78]. 12. Venter HJT and Verhoeven RL (1997) A tribal classification of the Conclusion Periplocoideae (). Taxon 46: 705-720. 13. Venter HJT, Verhoeven RL (2001) Diversity and relationships within the Decalepis is one of the most important woody medicinal climbing Periplocoideae (Apocynaceae). Ann Missouri Bot Gard 88: 550-568. genera of the family “Periploaceace”. Until 1997 it remains the 14. Ionta GM and Judd WS (2007) Phylogenetic relationships in Periplocoideae monotypic genera of this family, but now there are five species of (Apocynaceae s.l.) and insights into the origin of pollinia. Ann Missouri Bot Decalepis viz., D. hamiltonii, D. arayalpathra, D. salicifolia, D. khasiana Gard 94: 360-375. and D. nervosa which are endemic to Eastern and Western Ghats 15. Meve U, Liede S (2001) Reconsideration of the status of , of India. Amongst, D. hamiltonii is the most widespread and most and Notechidnopsis (-Ceropegieae). South utilized species. Except D. nervosa, roots are highly medicinal due African J Bot 67: 161-168. to the presence of vallinin-like fragrant compound namely, 2H4MB. 16. Holmgren PK, Holmgren NH and Barnett LC (1990) Index Herbarium. Part Ruthless collection of the roots is the main reason for the depletion I: The Herbaria of the World. New York Botanical Garden Press, New York. of these species in nature and urgently calls for their conservation 17. Vedavathy S (2004) Decalepis hamiltonii Wight & Arn.-an endangered source in natural habitats. Significant works have been carried out for the of indigenous health drink. Nat Product Rad 3: 22-23. conservation of D. hamiltonii through plant tissue culture. While, 18. Raju AJS, Ramana KV (2009) Pollination and seedling ecology of Decalepis only four reports are available for the D. araylpathra using plant tissue hamiltonii Wight & Arn. (Periplocaceae), a commercially important endemic culture. So, there is a need to exploit all the possible valuable strategies and endangered species. J Threatened Taxa 1: 497-506. of plant tissue culture like micropropagation, synseed preparation, 19. Anandalakshmi R, Prakash MS (2009). Seed germination and storage genetic transformation and elicitation for other species of Decalepis. characteristics of Decalepis hamiltonii implications for regeneration. Forest It is expected that the present article would provide an informative Tree Livelihood 19: 399-407. baseline to the researchers working on Decalepis species. 20. Saini RK, Giridhar P (2012) Effect of temperature, chemical treatment, hydration and plant growth regulators on germination of seeds and immature Acknowledgement zygotic embryo of swallow root (Decalepis hamiltonii). Research J Agri Sci 3: 40-44. Dr. Shiwali Sharma is thankful to DST, for the award of Young 21. Murti PB, Seshadri TR (1941) A study of the chemical components of the Scientist under Fast Track Scheme, SERB (vide no. SB/FT/LS- roots of Decalepis hamiltonii (Makali veru), Part I- Chemical composition of 364/2012) for providing research assistance. Dr. Anwar Shahzad the root. Proc Ind Acad Sci A 13: 221-232. gratefully acknowledges the financial support provided by UGC and 22. Murti PB and Seshadri TR (1941) A study of the chemical components of the UP-CST in the form of research projects (vide no. 39-369/2010 SR roots of Decalepis hamiltonii (Makali veru), Part II- A note on the preparation and vide no. CST/D3836). of inositol by solvent extraction. 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Citation: Sharma S, Shahzad A. An Overview on Decalepis: A Genus of Woody Medicinal Climbers. J Plant Sci Res. 2014;1(1): 104. 013